Manganese-Oxo Clusters as Contrast Agents for Magnetic Resonance Imaging

ABSTRACT

Nanoparticles for use as magnetic resonance imaging contrast agents are described. The nanoparticles are made up of a polymeric support and a manganese-oxo or manganses-iron-oxo cluster having magnetic properties suitable of a contrast agent. The manganese-oxo clusters may be Mn-12 clusters, which have known characteristics of a single molecule magnet. The polymer support may form a core particle which is coated by the clusters, or the clusters may be dispersed within the polymeric agent.

STATEMENT OF PRIORITY

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/196,725, filed Oct. 21, 2008 the disclosure of which is herebyincorporated by reference herein.

STATEMENT OF GOVERNMENT FUNDING

This work was supported in part by grants DMR-0304273 and CHE-05522586from the National Science Foundation. The U.S. government has certainrights in this invention.

FIELD OF THE INVENTION

The present invention relates to contrast agents for enhancing magneticresonance imaging. The agents of the present invention are particlescontaining metal-oxo clusters.

BACKGROUND OF THE INVENTION

Magnetic resonance imaging (MRI) has become a widely used tool formedical imaging and research. As a medical imaging technique, MRI ispreferred by patients as it does not require exposure to ionizingradiation. Further, MRI has proven to be the best way to obtain imagesof soft tissues in the body. Because MRI can be used for imaging softtissues, it can be effectively used to determine if a tumor or otherlesion has developed in an organ and to determine structural changes inthe brain, along with a multitude of other uses.

Very briefly. MRI requires placing the patient to be imaged in a strongmagnetic field. The magnetic field causes the hydrogen nuclei to alignso that they are spinning either parallel or antiparallel to themagnetic field. A radiofrequency (RF) pulse is then applied that excitesthe spinning hydrogen nuclei out of alignment. After application of theRF pulse, the excited nuclei relax back to alignment with the magneticfield, emitting RF signals that are detected by the MRI apparatus.Detection of the RF signals is used to calculate both the longitudinalrelaxation time (T₁) and transverse relaxation time (T₂), which are usedto form images of the patient.

The primary limitation of MRI is its sensitivity. Contrast agents areoften administered to the patient before imaging overcome sensitivitylimitations. These contrast agents cause changes in T₁ and T₂ forsurrounding hydrogen nuclei, which helps clearly differentiatestructures in the MRI image.

One class of contrast agents is the positive contrast agents, whichdecrease T₁ and T₂ to a similar extent and are typically used inobtaining T₁ weighted images. Positive contrast agents cause a tissuestructure associated with the contrast agent to appear brighter on anMRI image, making these agents very useful for increasing MRIspecificity.

A majority of the positive contrast agents approved by the Food and DrugAdministration (FDA) contain the paramagnetic metal ion gadolinium.Although free Gd³⁺ is toxic, it can be ligand protected to greatlyreduce its bioavailability. While Gd containing contrast agents aregenerally considered safe for administration to humans, recentlyconcerns have arisen due to the possible causal connection between Gdcontrast agents and the formation of nephrogenic systemic fibrosis inpatients with renal disfunction. Because of this, physicians are nowdebating the risks of administering Gd contrast agents to certain typesof patients, and the FDA has requested the manufacturers of Gd constrastagents to add new warning information to their labels (H. Steen and V.Schwenger, Pediatr Nephrol, 2007, 22, 1239;www.fda.gov/cder/drug/infopage/gcca/qa_(—)200705.htm).

There is also an environmental concern with the use of Gd contrastagents, as their impact on the environment after excretion by thepatient is largely unknown. As the popularity of MRI continues toincrease, more and more Gd contrast agents will be used, meaning thatmore Gd will be released into the environment.

Because of the possible health and environmental concerns related to theuse of Gd contrast agents, it is desirable to develop contrast agentsusing metals that are better tolerated both by the body and theenvironment. One metal that has paramagnetic properties making itsuitable for use as an MRI contrast agent while also being more benignto health and the environment is manganese. However, the toxicity offree Mn metal has limited the development of Mn contrast agents (A.Koretsky and A. Silva, NMR in Biomedicine, 2004, 17, 527). A Mn contrastagent, manganese dipyrodoxaldiphosphate (Mn-DPDP), has been approved bythe FDA as safe for human use and is sold by GE Healthcare under thename Teslascan™. However, Mn-DPDP has only been approved for imaging ofthe liver and has shown only limited effect on T₁ and T₂ in tissuesoutside of liver and kidney (G. Elizondo et al., Radiology, 1991, 178,73).

U.S. Pat. Nos. 5,330,742 and 5,548,870 to Deutsch et al., describeparamagnetic metal cluster compounds for enhancing MRI. However, thereduced metal cluster described, Z⁺[Mn₁₂X₁₂(OYR)₁₆(L)₄], is highlyreactive and has not been demonstrated to be stable in biologicalsystems because it would react immediately in water. Also, as thiscluster is insoluble in water, it must be conjugated to a carrier inorder to be delivered as a contrast agent. Although the patents describeconjugating this reactive cluster to polymeric or microspheric carriers,liposome carries and hydroxyapatite carriers, these carriers onlyprovide for limited increases in the solubility of the cluster. Further,the conjugation of the Z⁺[Mn₁₂X₁₂(OYR)₁₆(L)₄] cluster is unlikely tomake it more suitable for use in a biological system.

U.S. Pat. No. 5,364,953 to Beaty et al. describes paramagnetic metalclusters having oxygen and/or nitrogen containing ligands for use as MRIcontrast agents. However, the metal clusters taught in the patent arenot more than sparingly soluble in water and precipitate from solutionover time.

As such, there remains a need in the art for a highly water soluble andbio-stable contrast agent that provides high specificity while alsobeing benign to the body and the environment.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide contrast agents formagnetic resonance imaging having nanoparticles made up of a polymersupport and a manganese-oxo cluster. The manganese-oxo cluster maycontain from 2 to 12 manganese ions, and may have Mn(III) and/or Mn(IV)ions.

It is a further object of the present invention to provide contrastagents for magnetic resonance imaging having nanoparticles that containmanganese-oxo clusters of the general formula [Mn₁₂O₁₂(O₂CR)₁₆(H₂O)₄],where R may be an alkyl, vinyl, halo, oxy, or oxyalkyl group.

It is a further object of the present invention to provide contrastagents for magnetic resonance imaging having nanoparticles that containmanganese-iron-oxo clusters of the general formula[Mn₈Fe₄(O₂R)₁₆(H₂O)₄], where R may be an alkyl, vinyl, halo, oxy, oroxyalkyl group.

It is a further object of the present invention to provide contrastagents for magnetic resonance imaging having nanoparticles that containmanganese-iron-oxo clusters of the general formula[Mn₈Fe₄O₁₂(O₂R)₁₆(H₂O)₄], where R may be an alkyl, vinyl, halo, oxy, oroxyalkyl group.

It is a further object of the present invention to provide contrastagents for magnetic resonance imaging having a polymer with a monomericunit of the general formula:

where R is an organic side chain, and L is a carboxylic acid.

It is a still further object of the present invention to provide amethod for making a contrast agent for magnetic resonance imaging havingthe steps of: providing a first solution of a polymeric nano-bead havingcarboxylate groups on its surface in an alcohol; providing a secondsolution of a manganese-oxo compound of the general formula I or amanganese-iron-oxo compound of the general formulas II or III in thealcohol:

[Mn₁₂O₁₂(O₂CR)₁₆(H₂O)₄]  I;

[Mn₈Fe₄(O₂R)₁₆(H₂O)₄]  II;

[Mn₈Fe₄O₁₂(O₂R)₁₆(H₂O)₄],  III;

where R may be an alkyl, vinyl, halo, oxy, or oxyalkyl; mixing the firstsolution and the second solution; stirring the mixture for an amount oftime sufficient for substantially all of the carboxylate groups to reactwith the manganese-oxo compound; and isolating the resultant polymericbeads covered in a layer of manganese-oxo compound.

It is yet a further object of the present invention to provide a methodfor making a contrast agent for magnetic resonance imaging having thesteps of: providing a first solution containing a compound of thegeneral formula IV:

-   -   where L may be an organic side chain; and where R may be any        carboxylic acid; providing a second solution containing a        compound of the general formula V:

mixing the first solution with the second solution; adding apolymerization initiation agent; and reacting the mixture for an amountof time sufficient to cause completion of the reaction to form a polymerhaving a monomeric unit of the general formula VI:

L may be an organic side chain; and where R may be any carboxylic acid.

The present invention provides a method to obtain magnetic resonanceimages of cells, tissues, animals and human subjects. It is yet afurther object of the present invention to provide a method forobtaining a magnetic resonance image of a subject by administering tothe subject the a sufficient amount of the contrast agents of thepresent invention in order to obtain a magnetic resonance image with thedesired contrast, allowing a sufficient amount of time for the contrastagent to migrate throughout the subject, and obtaining a magneticresonance image of the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the attachment of Mn-12 to carboxylatecoated nano-beads. The nano-beads are shown in black and the Mn-12 ingray.

FIG. 2 shows a Fourier Transform Infrared (FTIR) spectrograph ofprecipitate formed from Mn₈O₁₂(Ac)₁₆ in aqueous solution. Althoughspecific material was determined, the Mn—O, O—H and COOH peaks areclearly visible.

FIG. 3 shows an FTIR spectrograph of Mn-12 re-isolated from acetic acidafter soaking for 24 hours. The cluster stretch from 450-700 cm⁻¹ isparticularly indicative of the intact cluster.

FIG. 4 shows a graph of 1/T₁ (s⁻¹) versus concentration (mM⁻¹) for Mn-12acetate in D₂O and dilute acetic acid.

FIG. 5 shows a graph of 1/T₂ versus concentration for Mn-12 complex inacetic acid (at 500 MHz).

FIG. 6 shows a graph of 1/T₁ versus concentration for 209 nm Mn-12coated beads (at 300 MHz).

FIG. 7 shows a graph of 1/T₂ versus concentration for 209 nm Mn-12coated beads (at 300 MHz).

FIG. 8 shows a plot of the per cluster r₁ (mM⁻¹s⁻¹) versus bead diameter(nm).

FIG. 9 shows a plot of Ln[intact Mn-12] as a function of time for Mn-12coated 209 nm beads.

FIG. 10 shows a plot of the AC susceptibility χ″-versus-temperature for[Mn₁₂O₁₂(VBA)16].

FIG. 11 shows a plot of χ′T-versus-Temperature (K) for [Mn₁₂O₁₂(VBA)16].

FIG. 12 shows an Arrhenius Plot for [Mn₁₂O₁₂(VBA)16].

FIG. 13 shows the dynamic light scattering of co-polymer beads.

FIG. 14 shows a scanning electron micrograph of co-polymer beads.

FIG. 15 shows an FTIR spectrograph of co-polymer beads. * indicatespeaks that match that of polystyrene; • indicates peaks that match thatof Mn₁₂O₁₂VBA₁₆.

FIG. 16 shows a plot of a thermogravimetric analysis of co-polymerbeads. 9.6% by weight remains at 1000° C., which is Mn₃O₄ as shown bypowder diffraction.

FIG. 17 shows the x-ray powder diffraction spectra of material remainingafter thermogravimetric analysis of co-polymer beads. * indicates peaksthat index to Mn₃O₄ and • indicates peaks that index to Mn₂O₃.

FIG. 18 shows a chromatogram of gel permeation chromatography performedon co-polymer beads.

FIG. 19 shows a plot of the AC susceptibility χ″-versus-temperature forco-polymer.

FIG. 20 shows an FTIR spectrograph of Mn₈Fe₄ as prepared in Example 2.

FIG. 21 shows the powder X-ray diffraction data, both actual andcalculated, for Mn₈Fe₄.

FIG. 22 shows plots of 1/T₁ (22A) or 1/T₂ (22B) versus concentration ofMn₈Fe₄.

FIG. 23 shows MRI imagining of Mn₈Fe₄ clusters in agar as is describedin Example 2.

FIG. 24 shows a plot of % viable prostate cancer cells detected in atryptan blue assay following exposure to various concentrations ofMn₈Fe₄ as is described in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

Nanoparticles containing manganese-oxo clusters for use as MRI contrastagents are described herein. In one embodiment, the nanoparticles of thepresent invention have a polymer support with Mn-oxo or Mn—Fe-oxoclusters attached to the support. The metal-oxo clusters used in theforming the nanoparticles of the present invention have magneticproperties that make them suitable for use as MRI contrast agents. Incertain embodiments, the nanoparticles of the present invention are fromabout 5 to about 1000 nm, about 5 to about 500 nm, about 5 to about 100nm, about 5 to about 50 nm in diameter.

In certain embodiments of the invention, the Mn-oxo clusters are Mn-12clusters. The Mn-12 clusters of the present invention may have a generalformula [Mn₁₂O₁₂(O₂CR)₁₆(H₂O)₄], where R may be an alkyl, vinyl, halo,oxy, oxyalkyl group. Mn-12 is considered the prototypical ‘SingleMolecule Magnet’ (SMM) (T. L is, Acta Crystallogr. Sect. B. 1980, 36,2042; A. Barra et al., J. Am. Chem. Soc, 1991, 113, 5873; R. Sessoli etal. Nature, 1993, 365, 141), meaning that the Mn-12 cluster acts as ananoscale individual magnet. The high spin state (S=10) and anisotropyof Mn-12 clusters results in striking magnetic properties (G. Christouet al., MRS Bulletin, 2000, Nov., 66). The clusters are typically mixedvalent and are made up of a tetrahedron of Mn (IV) ions, surrounded byeight Mn (III) ions, although other combinations of valencies may alsobe used in the present invention. In certain embodiments of theinvention, the Mn-12 cluster is [Mn₁₂O₁₂(O₂CCH₃)₁₆(H₂O)₄]. In otherembodiments of the invention, the Mn-12 cluster is[Mn₁₂O₁₂(O₂CC₆H₄CH═CH₂)₁₆(H₂O)₄].

Mn-12 clusters have properties which make them ideal as MRI contrastagents. The clusters have a very high spin state (high paramagneticsusceptibility), they are easily derivatized with a variety of ligands,and they have 4 waters that are intimately associated with the clustersthat are exchanged rapidly on an MRI timescale, because of theircoordination with labile Mn (III) ions (H. Eppley et al., J. Am. Chem.Soc., 1995, 117, 301). However, Mn-12 clusters by themselves have poorwater solubility, forming a flocculent material within minutes upondissolution in water. The present invention overcomes the solubilityproblems of Mil-12 by incorporating Mn-12 into nanoparticles that aresoluble in water.

In other embodiments of the invention, other Mn-oxo clusters may beused. For example, clusters having 2-12 Mn ions may be used inembodiments of the present invention. The clusters may be made up ofeither Mn(UI) or Mn(IV) ions, or may have both Mn(III) and Mn(IV) ionsin the same cluster. Examples of other Mn-oxo clusters that arecontemplated for use in the present invention include tetranuclearMn-oxo clusters described by Dube et al. (J. Am. Chem. Soc, 1998, 120,3704) and Chen et al. (Inorg. Chem., 2005, 44, 9567).

In other embodiments of the present invention, the metal-oxo clustersused are Mn—Fe-oxo clusters, which have both manganese and iron centers.The Mn—Fe-oxo clusters of the present invention may have a generalformula [Mn₈Fe₄(O₂R)₁₆(H₂O)₄], where R may be an alkyl, vinyl, halo,oxy, or oxyalkyl group. In certain embodiments, the Mn—Fe-oxo clustersof the present invention may have general formula[Mn₈Fe₄O₁₂(O₂R)₁₆(H₂O)₄], where R may be an alkyl, vinyl, halo, oxy, oroxyalkyl group.

It is also contemplated that Mn—Fe-oxo clusters having other ratios ofmanganese to iron centers may be used in the present invention. TheMn—Fe-oxo clusters, when conjugated as described herein, are watersoluble, stable and show significant relaxivity. In certain embodimentsof the invention, the Mn—Fe-oxo cluster is [Mn₈Fe₄(O₂CH₃)₁₆(H₂O)₄]. Incertain embodiments of the invention, the Mn—Fe-oxo cluster is[Mn₈Fe₄O₁₂(O₂CCH₃)₁₆(H₂O)₄].

It is further contemplated that the clusters of the present inventionmay exist in solvaled form, such as in water solvated form, acetic acidsolvaled form or in other solvate forms well known in the art. As anon-limiting example, an embodiment of the invention includes thesolvated Mn—Fe-oxo cluster [Mn₈Fe₄(O₂CH₃)₁₆(H₂O)₄].4H₂O.2CH₃COOH. In aspecific embodiment, the solvated Mn—Fe-oxo cluster has the formula[Mn₈Fe₄O₁₂(O₂CCH₃)16(H₂O)₄].4H₂O.2CH₃COOH.

In certain embodiments of the invention, the polymer support is apre-formed polymer bead. The polymer bead may be made of styrene, vinylbenzoic acid, vinyl alcohol, latex, or co-polymers thereof. In certainembodiments, the pre-formed polymer beads are surface coated with acarboxylate or other functional group that allows for facile attachmentof the Mn-oxo clusters. In one embodiment of the present invention, thepre-formed polymer beads are Polybead® Carboxylate Microspheres sold byPolysciences, Inc. of Warrington, Pa. In other embodiments, poroussilicon nanoparticles may be used, such as Mobil Composition of Matter41 (MCM-41), available from ExxonMobil Chemical Company of Houston. Tex.MCM-41 nanoparticle are useful carriers as they are benign in the bodyand have a high surface area for attaching clusters. Further, the MCM-41nanoparticles seem to enhance to relaxivity effects of the clusters.

If carboxylate coated polymer beads are used, the metal-oxo clusters maybe attached to the beads using the method described by Steckel et al.(Nano Lett., 2004, 4, 399), which is hereby incorporated by referenceherein. A general schematic of this procedure is shown in FIG. 1.Generally, a solution of carboxylate coated polymer beads in ethanol ismixed with a solution of metal-oxo cluster in ethanol. The mixture isallowed to stir for a period of time sufficient to cause the metal-oxoclusters to coat the polymer beads. The metal-oxo coated nanoparticlesare then isolated and washed, at which point they are ready to be madeinto a preparation for administration to a patient. Further descriptionsof forming metal-oxo coated polymer beads are shown in the examplesbelow.

In certain other embodiments of the present invention, the polymersupport is formed as a co-polymer with the metal-oxo clusters. Theco-polymers may be made with one or more of styrene, vinyl benzoic acid,vinyl alcohol or latex. The co-polymers may be formed into nanoparticlesduring polymerization or may be formed into a nanoparticle structureafter polymerization is complete.

In one embodiment of the present invention, the co-polymer is formedfrom Mn₁₂ vinyl benzoic acid (Mn₁₂-VBA) and styrene as shown in SchemeI, where R is an organic side chain and L is O₂CH═CH—R or any carboxylicacid.

In forming co-polymers, a wide range of organic side chains can be used,as are well known in the art, including, but not-limited to, alkanes,alkenes, alkynes, alcohols, halogens, ketones, aldehydes, carboxylicacids and ethers. In certain embodiments of the present invention, R maybe a C₁-C₆ alkane.

After the co-polymers are formed into nanoparticles, isolated andwashed, they are ready to be made into a preparation for administrationto the patient. Further descriptions of forming co-polymers can be foundin the examples below.

In certain embodiments of the invention, the nanoparticles are formed sothat free manganese is not released from the particles into solution.The nanoparticles may stay intact so that they do not release freemanganese until after they are excreted from the body.

In certain embodiments of the present invention, the nanoparticles maybe modified on their surface. Such modifications may be used tospecifically target the nanoparticles to certain areas of the body. Forexample, the nanoparticles may be modified with peptides, sugars,antibodies, ligands, dendrimers, nucleic acids or synthetic ligands thatcause the nanoparticle to be transported to or to remain in a higherconcentration in a specific tissue structure. The nanoparticles may alsobe modified with chemical groups that increase or decrease theirsolubility as needed.

Once metal-oxo nanoparticles are synthesized, they can be made into apreparation for administration to the patient to be imaged. In certainembodiments of the invention, the nanoparticles are suspended into asolution suitable for intravenous administration. Such a solution may bea suspension in pure water, or the solution may contain salts or othercompounds which are suitable for intravenous administration. Ifintravenous administration is used, the nanoparticle preparation may beadministered all at once as a bolus, or may be administered through anintravenous drip over a period of minutes to hours. In other embodimentsof the invention, the nanoparticles may be formulated into preparationssuitable for oral administration.

Once the nanoparticles of the present invention have been administeredto the patient in a sufficient amount and a sufficient amount of timehas passed for the nanoparticles to migrate to the tissues desired to beimaged, an MRI can be performed on the patient as is well known in theart. In certain embodiments, T₁ weighted images may be obtained,although it is also contemplated that T₂ and T₃* weighted images mayalso be obtained.

The nanoparticles and methods of the present invention may be used withany type of tissue that is suitable for imaging by MRI. In certainembodiments, the type of tissue may be organs, glands, nodes, connectivetissues, muscle tissues, nervous tissues, epithelial tissues, bones,tumors, both malignant and non-malignant, and other growths. Thenanoparticles and methods of the present invention may also be used witha single cell or group of cells derived from any tissue suitable forimaging by MRI.

The patients, or subjects in which the nanoparticles and methods of thepresent inventions can be used are preferably humans. However, it isalso contemplated that the nanoparticles and methods of the presentinvention may be used in any animal for which MRI can be performed.These subjects include mammals, such as domestic animals, veterinaryanimals, research animals and livestock. Specific examples include, butare not limited to dogs, cats, horses, mice, rats, hamsters, gerbils,apes, monkeys, rabbits, cattle, pigs and poultry such as chickens andturkeys.

The nanoparticles and methods of the present invention may also be usedin the imaging of tissues or cells in isolation. The tissues and cellsto be imaged may have been removed from a subject through surgery,biopsy or other procedure. The tissues and cells to be imaged may alsohave been grown in culture as is well known in the art. Along with beinguseful for medical diagnostic imaging of tissues and cells, thenanoparticles and methods of the present invention may be used forresearch purposes.

In specific embodiments, the methods of the present invention involveadding a sufficient amount of a contrast agent described herein to acell or tissue in order to acquire a magnetic resonance image. In otherembodiments, the methods involve administering a sufficient amount of acontrast agent described herein to an animal in order to acquire amagnetic resonance image.

The description of the present invention set forth herein, including thedrawings and the examples set forth below, is meant to providenon-limiting description of the compositions and methods of the presentinvention. It should be apparent that there are variations of thepresent invention not explicitly presented in this specification thatfall within the scope and the spirit of the invention as claimed.

EXAMPLES Example 1 Mn-12 Clusters Materials and Methods

4-vinylbenzoic acid (97%), Divinylbenzene (80%), Dichloromethane(anhydrous, 99%), Heptane (anhydrous, 99%), Ethanol (anhydrous, 99%,Toluene (reagent grade), Tetrahydrofuran (anhydrous, 99%),2,2′-Azobis(2-methylpropionitrile) (98%) and styrene were purchased fromSigma-Aldrich (St. Louis, Mo.). Sodium Dodecyl Sulfate was purchasedfrom Fluka (Seelze, Germany). Hexadecane was purchased from Alfa Aesar(Ward Hills, Mass.). Hydrochloric acid (37%, ACS grade) was purchasedfrom EMD Chemicals (San Diego, Calif.). Polybead® CarboxylateMicrospheres (2.73% Solids-Late) were purchased from Polysciences, Inc.(Warrington, Pa.) and separated from water by centrifugation at 4000 RPMfor 10 minutes and were allowed to dry overnight. Mn₁₂O₁₂(O₂CCH₃)₁₆4H₂Owas synthesized in our lab according to the literature. (T. L is, ActaCrystallogr. Sect. B, 1980, 36, 2042).

Fourier Transform Infrared Spectroscopy (FTIR) Characterization

FTIR experiments were recorded in the range 4,000-450 cm⁻¹, from pressedpellets in KBr on a Nicolet FTIR. UV-visible spectroscopy was measuredfrom 200-800 nm in ethanol on a HP UV-visible spectrometer in quartzcuvettes. Simultaneous TGA-DTA data were studied from samples in analuminium pan from 20-1000° C. with a heating rate of 10° C./min. GPCdata was obtained at a flow rate of 1 mL/min using a Hewlett Packardseries II 1090 Liquid Chromatograph and a Perkin Elmer LC-9S UV/VisSpectrophotometer as the detector. Samples were prepared for GPC byadding the solid to toluene (4 mL, 2 mg/mL) and mixing with an aqueoussolution of HCl (37%, 4 mL). This solution is stirred overnight at roomtemperature. After the reaction, the two layers are separated and thepolystyrene/toluene layer is pumped dry, giving the solid polystyrene,which is then dissolved in THF (1 mg/mL) to make the GPC solutions.

Atomic absorption was measured using a BUCK Scientific Model 200A AtomicAbsorption Spectrophotometer. Dynamic Light Scattering was used tocalculate particle size. In apparatus used, light from a HeNe laserilluminates dilute suspensions of particles. Light scattered at a fixedangle (usually 90°) is coupled though a narrow band pass optical filterinto a single mode optical fiber, which leads to a high sensitivityavalanche photodiode photon counting module (EG&G SPCM-15). The countrates from this detector are analyzed by a hardware autocorrelator(ALV-5000, ALV GmbH, Germany). Using standard assumptions, it can beshown that the decay rate of the count rate autocorrelation function isinversely proportional to the particle diffusion coefficient, from whichinformation on the particle size is obtained. Initial calculations ofthe particle sizes were determined using a single exponential fit to theautocorrelation functions. AC magnetic measurements were collected on aQuantum Design Physical Measurement System, in zero DC field fortemperatures ranging from 1.8-50 K. The AC frequency range was 10-10000Hz and the amplitude was 1-100e. Experimental data were corrected forsample holder and for diamagnetic contributions calculated from Pascalconstants.

Nuclear Magnetic Resonance (NMR) Experiments

Fresh samples for NMR measurements were prepared immediately prior touse. Both the cluster and cluster coated beads were dissolved indistilled water, and filtered via syringe filter. T₁ measurements weremeasured using the inversion recovery pulse sequence, in a field of 300MHz or 500 MHz, at room temperature with a least squares fit to 10 datapoints. The T₂ was obtained using a conventional spin echo sequence, ona 300 MHz spectrometer and using Carr-Purcell-Meiboom-Gill (CPMG)sequence on 500 MHz Bruker spectrometer. Relaxivity was determined fromthe slope of a plot of 1/T₁ or 1/T₂ versus concentration of Mn-12 andMn-12 coated beads. The cluster concentration was determined by atomicabsorption of Mn.

Concentration Dependent Effect of Mn-12 on T₁ and T₂

Based on simple criteria, Mn-12 appears ideal as a contrast agent. Ithas a very high spin-state (high paramagnetic susceptibility), is easilyderivatized with a variety of carboxylic acids, and has 4 waters thatare intimately associated with the cluster that are exchanged rapidly onthe NMR timescale (due to coordination to the labile MnIII) (H. Eppleyet al. J. Am. Chem. Soc, 1995, 117, 301). However, upon dissolution ofMn-12 in distilled water, within minutes a flocculent material forms. AnFTIR of this precipitate is shown in FIG. 2. This poor solubility is amajor barrier for potential biomedical applications. However, in thepresence of excess carboxylic acid. Mn-12 is stable in aqueous aceticacid solutions and can be re-isolated after many hours.

Taking advantage of this property of Mn-12, the concentration dependenteffect of Mn-12 on the T₁ and T₂ of aqueous protons in acetic acidsolutions was studied. The cluster was identified by FTIR (FIG. 3) aftereach NMR experiment to confirm the structural integrity, although below0.5 mM the amount of material recovered was so small definitivespectroscopic characterization was difficult. A graph of 1/T₁ versusconcentration (FIG. 4) was linear with R²=0.9978 and gave an r₁ of3.0±0.1 mM⁻¹V. The analogous graph for 1/T2 (FIG. 5), had an R²=0.9961and gave r₂=48±1 mM⁻¹s⁻¹. For comparison of our S=10 molecule togadolinium S=7/2 molecules, relaxivity values are reported in mM⁻¹ ofthe cluster (as opposed to mM⁻¹ of metal). Gadolinium complexestypically have r₁ values that range from 4-16 depending on the ligands,(S. Aime et al. J. Magn. Res. Imaging, 2002, 16, 394) while iron oxidenanoparticles are generally found to be −30 mM⁻¹s⁻¹ (r₂˜100 mM⁻¹s⁻¹)(Y.-X. J. Wang et al., Eur. Radiol., 2001, 11, 2319). Interestingly ther₂/r₁ values for iron oxide tend to range from 40-100, resulting in a T₂weighted contrast agent. Here the Mn-12 clusters appear to beintermediate, somewhat closer to Gd complexes, with a r₁/r₂ of 16.

The experiments reported here were predominantly done at much higherfields (300-500 MHz), than normally used for MRI, and the temperatureswere almost all room temperature. The relaxivity of Mn-12 at 37° C. wasmeasured, and it was found that the relaxivity decreased at highertemperatures, similar to molecules for which the rotational correlationtime is a factor (K. Raymond et al., Bioconjugate Chem. 2005, 16, 3).Although a thorough study of the field dependence of the relaxivity hasnot been done, measurements at 300, 400, and 500 MHz show that therelaxivity appears to increase with higher fields, similar to theGd—HOPO complexes and unlike commercially available contrast agents (K.Raymond et al., Bioconjugate Chem. 2005, 16, 3). This may be anadvantage as the fields used for MRI have been increasing, placing newdemands on contrast agents D. Fulton et al., Chem. Commun. 2006, 1064.

Synthesis of Mn-12 Coated Polystyrene Beads

It has been previously demonstrated that Mn-12 can undergo ligandexchange for attachment onto any surface containing carboxylic acidfunctionality (Steckel et al., Nano Lett., 2004, 4, 399). With multiplesites of attachment, the chelate effect stabilizes the cluster onsurfaces even under a flow of fresh solvent as demonstrated by quartzcrystal microbalance studies of carboxylate terminated SAMs with amonolayer of Mn-12 attached. As described herein, using a similar ligandexchange method, it is possible to attach the cluster to a polystyrenebead with a carboxylate surface, forming a water soluble, stable bead asillustrated in FIG. 1.

The Mn-12 coated polystyrene beads were synthesized as follows. Drypolystyrene beads were suspended in dry ethanol and soaked for 24 hoursprior to surface attachment. A solution of Mn₁₂O₁₂(O₂CCH₃)₁₆ in ethanol(2 mM) was filtered and added to the ethanolic solution of beads andthis mixture was stirred for 2 hours. The Mn-12 coated beads wereremoved from solution by centrifugation at 4000 RPM for 10 minutes. Thebeads were washed with ethanol and centrifuged and isolated 3 times. TheFTIR of the beads matched that of polystyrene. Based on monolayercoverage (assuming 177 ng/cm² from QCM data of thin films), thetheoretical [Mn] for 47 nm, 120 nm, 209 nm, 489 nm, 994 nm beads were7.0%, 3.1%, 1.9% and 0.83% and based on atomic absorption the [Mn] was1.7%, 2.3%, 0.5% and 0.2% respectively. The actual concentration of Mnby atomic absorption was used in determining the relaxivity reported.These were reproducible. The beads coated with Mn-12 can be dissolved inwater, and after 48 hours the supernatant shows no evidence of manganeseby atomic absorption experiments.

Plots of 1/T₁ and 1/T₂ versus concentration for 209 nm beads are shownin FIGS. 6 and 7, respectively. The relaxivity of cluster coated 200 nmbeads is included in Table 1. The n per molecule increased to 37.5±4.5mM⁻¹s⁻¹. The Solomon-Bloembergen-Morgan theory (N. Bloembergen et al.,Phys. Rev. 1948, 73(7), 679; N. Bloembergen et al, J. Chem. Phys. 1961,34(3), 842; I. Solomon, Phys. Rev. 1955, 99(2), 559) predicts that theinner sphere relaxivity should be dominantly influenced by thereorientational correlation time (τ_(R)). In essence, longer τ_(R) orslower tumbling agents should have faster relaxation rates and higherrelaxivities (E. Weiner et al., J. Am. Chem. Soc. 1996, 118, 7774; M.Bottrill et al., Chem. Soc. Rev. 2006, 35, 557). Although thereorientational correlation time has been associated with molecularweight, shape is also important, and the effect can be enhanced forspherical agents (M. Lowe, Aust. J. Chem. 2002, 55, 551; P. Caravan,Chem. Soc. Rev., 2006, 35, 512). For example, multimeric gadolinumcomplexes in modified dextran polymers the per gadolinium relaxivity r₁increases to 10.6 mM⁻¹s⁻¹ (C. Casali et al., Acad. Radiol. 1998, 5,S214). Other macromolecular gadolinium complexes, such as polyamidedendrimers, also exhibit an increased r₁ (˜16.5 mM⁻¹sec⁻¹ pergadolinium) (M. Rohrer et al., Invest. Radiol. 2005, 40, 715).

TABLE 1 Relaxivity of Clusters and Cluster Coated Beads Sample r₁ r₂r₂/r₁ Mn₁₂O₁₂Ac₁₆ RT [a] 3.0 ± 0.1 48 ± 1 16 Mn₁₂O₁₂Ac₁₆ (37° C.) [a]2.7 ± 0.1 34 ± 1 13 Mn₁₂O₁₂—OOC-Bead (RT) [b] 37.5 ± 4.5  2585 ± 74  69[a] experiment in D₂O and dilute acetic acid (500 MHz) [b] in D₂O, 209 ±11 nm bead (300 MHz).Effect of Bead Diameter on r₁

To determine whether the reorientational correlation time is importantin this system the effect of bead diameter on n was investigated. As thebead diameter was increased from 47.1±1.5 nm to 209±11 nm, the percluster r₁ increased from 22.0±2.5 s⁻¹ to 37.5±4.5 s⁻¹ as shown in FIG.8.

The relative stability of Mn-12 in neutral aqueous solutions uponsurface attachment was also studied. It was found that the T₁ slowlyincreased (i.e. the relaxivity decreased) over a 10 hour period.Solutions of cluster coated beads that have soaked over night exhibit noprecipitate and do not release manganese into the solution. Thereforegradual decrease in n over time for Mn-12 coated beads, must be a resultof a reduction of the spin state possibly due to structural changes tothe surface attached cluster. Assuming that the relaxivity of thecluster is a constant, the changes in T₁ were used to calculate thechange in concentration of intact cluster. This assumes that whateverform of manganese oxide is left on the surface is inactive towardsrelaxivity. A plot of ln[Intact Mn-12] as a function of time followedfirst order kinetics (FIG. 9) with a rate constant k=0.013 hr⁻¹, and ahalf life of 51 hours.

Formation of Co-polymers of Substituted Mn-12 Clusters and Styrene

To enhance the stability of the cluster further, an the alternative offorming co-polymers of styrene with substituted Mn-12 clusters wasinvestigated. Using ‘miniemulsions’, polymerization of droplets with asize range of 50-500 nm (K. Landfester, Annu. Rev. Mater. Res. 2006, 36,231; K. Landfeester et al., Macromolecules, 1999, 32, 5222), stable,homogenous, magnetic co-polymer nanobeads were formed. Ligandsubstitution of acetate for a polymerizable carboxylic acid such asmethacrylic acid results in a cluster with functionality for olefinpolymerization. The reaction with styrene, emphasizing thepolymerization chemistry, is shown in Scheme I, where L=O2CH═CH—R.

Similar bulk co-polymers have been reported for methacrylic acidsubstituted clusters polymerized with methyl methacrylate which resultedin materials with enhanced chemical stability towards water and heat (S.Willemin et al., New. J. Chem. 2004, 28, 919; F. Palcio et al., J.Mater. Chem. 2004, 14, 1873). Synthesis of a substituted cluster withL=vinyl benzoic acid is reported for the first time herein. The vinylbenzoic acid may be co-polymerized with styrene. The initial choice ofstyrene as the co-monomer was based on biostability, and the fact thatstyreneis inexpensive and easily surface functionalized to controlsolubility and the binding of antibodies or proteins (K. Landfester etal., J. Phys: Condensed Matter, 2003, 15, S1345) as well as based onprevious experience forming monodispersed nanobeads of polystyrene (E.Van Keuren, J. of Dispersion Sci. and Tech. 2004, 25(4), 1). Vinylbenzoic acid was chosen as the substituent ligand due to its similarityto styrene.

Mn-12 vinyl benzoic acid monomers (Mn-12-VBA) was prepared as follows.The preparation of Mn12-VBA was based on a method described elsewhere(a) E. Weiner et al., J. Am. Chem. Soc. 1996, 118, 7774; M. Bottrill etal., Chem. Soc. Rev.

2006, 35, 557). Briefly, 4-vinylbenzoic acid (1.5 mmol) was added to aslurry of Mn₁₂O₁₂(O₂CCH₃)₁₆ (0.0625 mmol) in dichloromethane (25 mL).The solution was stirred for 4 hours, filtered to a beaker and layeredwith heptanes (50 mL). A brown solid precipitate formed afterapproximately 24 hours. The procedure was repealed to ensure completeexchange of vinylbenzoic acid for acetate to yield a final productMn₁₂O₁₂(OOCC6H₄CH═CH₂)₁₆(H₂O)₄. IR (KBr cm⁻¹): 3401 (m, br), 3082 (w),3066 (w), 3006 (w), 2923 (w), 1929 (w), 1835 (w), 1700 (m), 1624 (w),1606 (s), 1578 (s), 1507 (s), 1410 (vs), 1347 (s), 1289 (m), 1183 (s),1110(m), 1016 (s), 988 (m), 914(m), 862 (s), 790 (s), 771 (m), 718 (s),663 (m), 625 (s), 552 (m), 518 (m). UV-visible 1_(max) acctonitrile nm(εMn, L⁻¹cm⁻¹): 510 nm (580), 747 nm (108). Anal. Calc. (Found) forC₁₅₃H₁₃₀O₅₁Mn₁₂: C, 52.77 (52.31); H, 3.70 (3.95). Percent yield 91.90%.

Before synthesizing and studying the co-polymers the magnetic propertiesof this new Mn-oxo cluster were investigated, Mn₁₂O₁₂(O₂C—C₆H₄—CH═CH₂)16([Mn₁₂O₁₂VBA₁₆]) because it has not been reported previously. Asdescribed below, the magnetic properties are generally consistent withMn-12 with acetate ligands. The ground state of the cluster has beendetermined using the in-phase molar AC susceptibility χ⁻¹ measurements.From the plateau in the χ′T versus T plot, an effective moment μeff for[Mn₁₂O₁₂VBA₁₆] is found at 19.1 μB with spin S=9.1, compared withμeff=17.4 μB and spin S=9 for [Mn₁₂O₁₂Ac₁₆] (E. Weiner et al., J. Am.Chem. Soc. 1996, 118, 7774; M. Bottrill et al., Chem. Soc. Rev. 2006,35, 557). The Weiss temperature, Θ/K=3.9, and the Curie constant wasC=31.3 were based on analysis of the Curie plot.

One of the most striking features of ‘Single Molecule Magnets’ is thetemperature dependence of the out-of-phase AC susceptibility signal(χ″). The peak corresponds to the temperature at which the rate of theflip of the molecular moment is equal to the AC frequency, v′ (E. Weineret al., J. Am. Chem. Soc. 1996, 118, 7774; M. Bottrill et al., Chem.Soc. Rev. 2006, 35, 557). The appearance of two maxima in χ″ as afunction of temperature at 2.2 and 4.4K at 10 Hz (see FIG. 10) suggeststwo relaxation mechanisms.

Several complexes with composition [Mn₁₂O₁₂(O₂CR)16] exhibit two out-ofphase AC magnetic susceptibility signals, typically in the 4-7K and 2-3Kregion. The presence of two relaxation mechanisms has been attributed tothe presence of two isomers of the complex, either in the placement ofthe four H₂O ligands or Jahn-Teller isomerism (S. Aubin et al., Inorg.Chem. 2001, 40, 2127). Likewise, as shown in FIG. 11, there are twoplateaus in the χ′T vs T, consistent with two relaxation mechanisms. Theshift to lower temperature as the frequency decreases in the χ″ as afunction off, is typically seen in Mn-12 clusters (H. Eppley et al., J.Am. Chem. Soc, 1995, 117, 301). As shown in FIG. 12, using an Arrheniusplot of 1/v⁻¹ versus the peak temperature (where v⁻¹ is the ACfrequency) the energy barrier to spin reversal. U/K was determined to be65.9 with a relaxation time, τ0=4.22×10⁻⁸ s⁻¹ (high temperature peak)and U/K=27.4 with a of 1.04×10⁻⁸s⁻¹ (low temperature peak). This iscomparable to the high temperature peak with U/K=61 and τ=2.1×10⁻⁷ s⁻¹for [Mn₁₂O₁₂Ac₁₆](R. Sessoli et al., Nature, 1993, 365, 141; D.Gatteschi et al., Science 1994, 265, 1054).

For co-polymerization, miniemulsions were prepared from [Mn₁₂O₁₂VBA₁₆],styrene, 1% divinylbenzene (DVB), the hydrophobe hexadecane in aqueoussolutions of SDS using AIBN as an initiator. For pure polystyreneminiemulsions, the hydrodynamic diameter of the beads formed isgenerally determined by the ultrasonication time, polymerizationinitiation time after sonification, as well as surfactant concentration(K. Landfester et al., Macromolecules, 1999, 32, 5222). Our preliminaryexperiments used ultrasonification times and surfactant concentrationsassociated with 100 nm polystyrene beads. However, analysis of dynamiclight scattering (DLS) experiments (FIG. 13) indicated that the medianradius of the beads were 1 μm, and SEM images (FIG. 14) suggestagglomeration of smaller particles. It is likely that the Mn-12 and DVBaffect the solubility, so the amount of surfactant was increasedsignificantly resulting in beads with much smaller diameters. Thereappeared to be two main components to the particle size distribution at40 nm and ˜1 μm in diameter from the DLS.

Miniemulsion polymerization was performed as follows. The substitutedcluster, Mn₁₂O₁₂(OOCC₆H₄CH═CH₂)₁₆(H₂O)₄ (0.0345 mmol), styrene (6.78mmol) and divinylbenzene (1 wt %) were combined first to form the oilphase. To this solution, both 2,2′-Azobisisobutyronitrile (0.24 mmol)and hexadecane (0.13 mmol) were added, stirred and degassed. A secondsolution, containing sodium dodecyl sulfate (0.28 mmol) dissolved indistilled H₂O (8 mL) forming the aqueous phase, was degassed thencombined with the Mn-12/styrene solution. The resulting mixture was thenplaced in an ultrasound (Fisher 550 Sonic Dismembrator) at a setting of5 for 90 seconds. Finally, the mixture was placed in a water bath at 60°C. (New Brunswick Scientific C76 Water Bath Shaker) for 6 hours. Thesolution was immediately removed from the water bath and theMn-12/polystyrene beads were removed from solution by centrifugation at4000 RPM. The beads were washed with ethanol, and dried. The manganesecontent based on atomic absorption was 0.82%, compared with thetheoretical amount (assuming a 1:200 ratio of cluster to styrene) of2.73%. Gel permeation chromatography of the organic fraction gave abroad peak at 15 minutes, which corresponded to the average molecularweight of 6217 g/mol. FIG. 15—FTIR (KBr, cm⁻¹): 3444 (m, br), 3082 (w),3061 (w), 3026 (w), 2958 (m), 2920 (s), 2850 (s), 1940 (w), 1600 (m),1581 (w), 1492 (m), 1454 (m), 1412 (s), 1261 (s), 1217 (s), 1182 (m),1095 (s), 1026 (m), 908 (m), 862 (w), 802 (s), 758 (m), 698 (vs), 626(w), 534 (m), 503 (m), 457 (m). As shown in FIG. 16, thermogravimetricanalysis of the beads exhibited three steps in the thermal decompositionat 128° C., 227° C. and 397° C., leaving 9.6% of the mass. As shown inFIG. 17, the X-ray powder diffraction pattern of the residue matchedthat of Mn₃O₄, d (hkl); 4.92 (101), 3.08 (112), 2.88 (200), 2.76 (103),2.49 (211), 2.04 (220), 1.70 (312), 1.64 (303), 1.54 (224), 1.44 (314).

Unlike miniemulsions of pure polystyrene, which polymerize between 10mins−2 hours depending on the conditions, the copolymer reactions wereleft for −6 hours. At shorter periods, it was possible to smell styreneand no powder was obtained. After 6 hours, the FTIR of the isolatedproduct matches that of polystyrene. The intensity of the C═C bands at1630 cm−1 were lost, suggesting complete polymerization. Unfortunatelythe peaks unique to the cluster (particularly the core stretches between450-650 cm−1) are obscured by die strong polymer peaks.

As seen previously in the literature for bulk co-polymers, the TGA data(FIG. 16) showed that the co-polymer beads decompose in one step, athigher temperatures (here by 7° C.) than pure polymer, and the residuehas an X-ray powder diffraction pattern that indexes to Mn₃O₄ (FIG. 17).The washed and dried co-polymer was separated into its organic andinorganic components with an acid-toluene solvent mixture. As shown inFIG. 18, the isolated organic polymer was then characterized by gelpermeation chromotography (GPC). The GPC indicated the average molecularweight was 6217 g/mole, which corresponded to ˜60 styrene units, quitelow compared with miniemulsion reactions of pure polystyrene (withmolecular weights as high as 105). The manganese content of theinorganic component, based on atomic absorption suggested a metal molarpercent of 0.82% Mn. Interestingly, this was higher than expected forthe reactant stoichiometry which would give 0.5% (for stoichiometricratio of 1:200).

The AC susceptibility measurements indicate the spin state of the metalwas 6, lower than that found for the unpolymerized cluster. This isconsistent with bulk measurements of the copolymer where Mii-12 issubstituted with acrylate, Mn₁₂O₁₂(Acrylate)16, and polymerized withethyl acrylate under similar conditions (S. Willemin et al. New J.Chem., 2004, 28, 919.) The other example of an Mn-12 copolymer,Mn12O12(Methaerylate)16 with methyl-methacrylate did not report the 1/χ′versus temperature or the spin state (F. Palacio et al. J. Mater. Chem.2004, 14, 1873.) Interestingly, a peak in χ″ versus temperature wasobserved, as show in FIG. 19. (S. Willemin et al. New J. Chem., 2004,28, 919; F. Palacio et al. J. Mater. Chem. 2004, 14, 1873.)

The spin state was calculated to be only S=5, much lower than expected.The energy barrier for the two peaks were 64.8 (U/K) and τ₀=1.29×10⁻⁹ Hzfor the high temperature peak, and 19.6 (U/K) and to =3.99×10−8Hz forthe low temperature peak.

Preliminary relaxivity measurements were done, and an estimate of r₁ was23.6±2.5 mM−1s−1. However it is noted that there was a wide distributionof particle sizes in the described synthesis, and as has beendemonstrated here, there is a strong dependence of relaxivity on beaddiameter.

Example 2 Mn—Fe-oxo Clusters General Information

All chemicals and solvents were obtained from Sigma-Aldrich and used asreceived. Infrared spectra were measured in the range 450-4000 cm⁻¹ aspressed KBr pellets on a Nicolet 380 FTIR spectrometer. Elementalanalysis was performed on a Perkin Elmer 2400 Elemental Analyzer, usingacetanilide as standard. X-ray powder diffraction patterns were obtainedusing a Rigaku RAPID Curved IP X-ray powder diffiactometer with Cu Kαradiation and an image plate detector.

Synthesis of Mn₈Fe₄

[Mn₈Fe₄O₁₂(O₂CCH₃)16(H₂O)₄].4H₂O.2CH₃COOH, denoted here as Mn₈Fe₄, wasprepared as described in previous reports (A. R. Schake, et al., InorgChem., 1994, 33, 6020-6028.; A R Schake, et al., J. Chem. Soc. Chem.Commun., 1992, 181-183.) Just briefly, KMnO₄ (6.4 mmol) was added to aslurry of Fe(O₂CCH₃)₂ (16.3 mmol) in 40 mL 60% (v/v) acetic acid/H₂O andslowly heated to 60° C. The resulting reddish-brown solution wassubsequently cooled to room temperature, layered with 40 mL acetone andallowed to stand undisturbed for several days. The resulting blackcrystals were collected via vacuum filtration, washed with acetone anddried. IR (cm⁻¹): 3441 (br), 1709 (m), 1585 (s), 1421 (s), 3349 (m), 714(s), 662 (m), 616 (m), 565 (m), 539 (m). Anal calc: C, 20.95; H, 3.52.Found: C, 21.56; H, 3.43. See FIG. 20. The powder X-ray diffractionpattern obtained from the synthesized Mn₈Fe₄ is shown in FIG. 21.

NMR Relaxation Studies

NMR relaxation data were obtained using a Bruker AM 300 MHz spectrometerinterfaced with a TecMag DSPect acquisition system. Fresh solutions ofMn₈Fe₄ in D₂O were prepared (ranging from 0.6 mM metal to 3 mM metal)immediately prior to use. T₁ measurements were recorded using theinversion recovery pulse sequence at room temperature with a leastsquares fit to 10 data points. On the other hand, T₂ values weremeasured using a conventional spin echo sequence. Relaxivity values, r₁and r₂, were determined from the slopes of plots of 1/T₁ (FIG. 22A) or1/T₂ (FIG. 22B), respectively, versus concentration of Mn₈Fe₄:

(1/T _(i))_(obs)=(1/T _(i))₀ +r _(i) [M]

where (1/T_(i))_(obs) is the relaxation rate in the presence of Mn₈Fe₄,(1/T_(i))₀ is the relaxation rate in the absence of Mn₈Fe₄, r_(i) is therelaxivity and [M] is the concentration of Mn₈Fe₄ in mM metal.

MRI In Vitro

Samples with Mn₈Fe₄ concentrations ranging from 0.6 mM metal to 2.4 mMmetal were prepared in 3% agar and loaded into phantoms. Imaging wasperformed on a 7T Bruker Biospin (Germany/USA) imaging console. Theprotocol used was a spin-echo fast imaging technique. TurboRARE-T2(ParaVision v.4.0 software), with the following set of parameters: echotime (TE) 36 ms, repetition time (TR) 4200 ms, image matrix 256×256,slice thickness 1.0 mm and field of view (FOV) of 8.00 cm. See FIG. 23.

Cytotoxicity Studies

The human prostate cancer derived cell line, DU-145, was used in thisstudy. Cells were split into a six-well plate containing cover slips andmaintained in cell culture media at 37° C. Four wells containing thecells were incubated with Mn₈Fe₄ (total concentration ranging from 0.006mM metal to 0.75 mM metal), one well was incubated with iron oxidenanoparticles (Bangs laboratories) for comparison, while another wellwas left as a control. After 24 hours, cells were trypsinized, followedby dilution (1:5) of a small sample of the cell suspension in 0.4% (w/v)trypan blue stain. The cells (viable and non-viable) were counted usinga hemacytometer. The percentage of the viable cells was calculated usingthe equation below:

${\% \mspace{14mu} {viability}} = {\frac{\# \mspace{14mu} {of}\mspace{14mu} {Viable}\mspace{14mu} {cells}\mspace{14mu} {counted}}{{Total}\mspace{14mu} {cells}\mspace{14mu} {counted}} \times 100}$

The results of the cytoxicity studies are plotted in FIG. 24.

1-33. (canceled)
 34. A contrast agent for magnetic resonance imagingcomprising: (1) nanoparticles, wherein said nanoparticles comprise: apolymer support; and a manganese-oxo cluster or a manganese-iron-oxocluster; or (2) a polymer having a monomeric unit of formula IV:

wherein L is a carboxylic acid; and wherein R is an organic group. 35.The contrast agent of claim 34, wherein the manganese-oxo clustercomprises between 2 and 12 manganese ions.
 36. The contrast agent ofclaim 34, wherein the manganese-oxo cluster comprises Mn(III) ions. 37.The contrast agent of claim 34, wherein the manganese-oxo clustercomprises Mn(IV) ions.
 38. The contrast agent of claim 34, wherein themanganese-oxo cluster is a compound of Formula I[Mn₁₂O₁₂(O₂CR)₁₆(H₂O)₄]  I; wherein R is selected from the groupconsisting of alkyl, vinyl, halo, oxy, and oxyalkyl.
 39. The contrastagent of claim 38, wherein R is methyl.
 40. The contrast agent of claim38, wherein R is C₆H₄CH═CH₂.
 41. The contrast agent of claim 34, whereinthe manganese-iron-oxo cluster is a compound of Formula II[Mn₈Fe₄(O₂R)₁₆(H₂O)₄]  II; wherein R is selected from the groupconsisting of alkyl, vinyl, halo, oxy, and oxyalkyl.
 42. The contrastagent of claim 41, wherein R is methyl.
 43. The contrast agent of claim41, wherein R is CCH₃.
 44. The contrast agent of claim 34, wherein themanganese-iron-oxo cluster is a compound of Formula III[Mn₈(Fe₄O₁₂(O₂R)₁₆(H₂O)₄]  III; wherein R is selected from the groupconsisting of alkyl, vinyl, halo, oxy, and oxyalkyl.
 45. The contrastagent of claim 44, wherein R is methyl.
 46. The contrast agent of claim44, wherein R is CCH₃.
 47. The contrast agent of claim 34, wherein thepolymer support is a polymeric bead.
 48. The contrast agent of claim 34,wherein is a polymer made up of monomers selected from the groupconsisting of: styrene, vinyl benzoic acid, vinyl alcohol, latex, andcombinations thereof.
 49. The contrast agent of claim 47, wherein thepolymeric bead is a polystyrene bead.
 50. The contrast agent of claim34, wherein the polymer support is a core structure having the clustersbonded to the surface of the core structure.
 51. The contrast agent ofclaim 34, wherein the clusters are dispersed throughout the polymersupport.
 52. The contrast agent of claim 34, wherein the nanoparticle iswater soluble.
 53. The contrast agent of claim 34, wherein L isO₂CH═CH—R′; and wherein R′ is selected from the group consisting ofalkanes, alkenes and alkynes.
 54. The contrast agent of claim 34,wherein R is C₁-C₆ alkyl.
 55. A method for synthesizing the contrastagent for magnetic resonance imaging of claim 34 comprising: providing afirst solution of a polymeric bead having carboxylate groups on itssurface in an alcohol; providing a second solution of a manganese-oxocompound of formula I or a manganese-iron-oxo compound of formula II orformula III in the alcohol:[Mn₁₂O₁₂(O₂CR)₁₆(H₂O)₄]  I;[Mn₈Fe₄(O₂R)₁₆(H₂O)₄]  II;[Mn₈(Fe₄O₁₂(O₂R)₁₆(H₂O)₄]  III; wherein R is selected from the groupconsisting of alkyl, vinyl, halo, oxy, and oxyalkyl; mixing the firstsolution and the second solution; stirring the mixture for an amount oftime sufficient for substantially all of the carboxylate groups to reactwith the manganese-oxo or manganese-iron-oxo compound; and isolating theresultant polymeric beads covered in a layer of manganese-oxo ormanganese-iron-oxo compound; or providing a first solution containing acompound of Formula V:

wherein L is a carboxylic acid; and wherein R is an organic group;providing a second solution containing a compound of Formula VI:

mixing the first solution with the second solution; adding apolymerization initiation agent; and reacting the mixture for an amountof time sufficient to cause completion of the reaction to form a polymerhaving a monomeric unit of Formula IV:

wherein L is a carboxylic acid; and wherein R is an organic group. 56.The method of claim 55, wherein the alcohol is ethanol.
 57. The methodof claim 55, wherein R is methyl.
 58. A method for obtaining a magneticresonance image of a subject comprising: administering to the subjectthe a sufficient amount of the contrast agent of claim 34 in order toobtain a magnetic resonance image with the desired contrast; allowing asufficient amount of time for the contrast agent to migrate throughoutthe subject; and obtaining a magnetic resonance image of the subject.59. The method of claim 58, wherein the contrast agent is administeredintravenously.
 60. The method of claim 58, wherein the magneticresonance image obtained is a T₁ weighted image.
 61. The method of claim58, wherein the magnetic resonance image obtained is a T₂ weightedimage.
 62. The method of claim 58, wherein the magnetic resonance imageobtained is a T₂ weighted image.